Molecular-modeling based design, synthesis, and activity of substituted piperidines as gamma-secretase inhibitors

Alzheimer's disease (AD) is a debilitating disease widely thought to be associated with the accumulation of beta amyloid (Abeta) in the brain. Inhibition of gamma-secretase, one of the enzymes responsible for Abeta production, may be a useful strategy for the treatment of AD. Described below is a series of gamma-secretase inhibitors designed from a scaffold identified by a ROCS [J. Comput. Chem.1996, 17, 1653] search of the corporate database.     

Nanoelectropulse-induced phosphatidylserine translocation

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis-caspase activation and loss of mitochondrial membrane potential-appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000 Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.     

Description of a new species of Phylloscopus warbler from Vietnam and Laos

A new species of Phylloscopus warbler, which we name Phylloscopus calciatilis Limestone Leaf Warbler, is described from central and northern Vietnam and central and northern Laos; it probably also breeds in southernmost China. In morphology, the new species is very similar to Sulphur-breasted Warbler Phylloscopus ricketti , but it is smaller with a proportionately larger bill and rounder wing. Its song and calls are diagnostic. Based on mitochondrial and nuclear DNA, the new species is most closely related to P. ricketti and Yellow-vented Warbler Phylloscopus cantator , and it is inferred to be sister to the latter. The mitochondrial divergences between these three species are at the low end of the variation found in other species of Phylloscopus and Seicercus warblers, but greater than in other taxa generally treated as subspecies. Possible introgressive hybridization between the new species and P. ricketti is discussed, but more data are needed to establish whether it does occur and, if it does, to what extent. The new species appears to have a restricted breeding range in limestone karst environments, where it is locally common and therefore not under any immediate threat. In view of the recognition of the new species, all previous records of P. ricketti sensu lato need to be re-evaluated.     

No change in myonuclear number during muscle unloading and reloading

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

Variable agronomic practices, cultivar, strain source and initial contamination dose differentially affect survival of Escherichia coli on spinach

Aims: Greenhouse and field trials were conducted under different agronomic practices and inoculum doses of environmental Escherichia coli and attenuated E. coli O157:H7, to comparatively determine whether these factors influence their survival on leaves and within the rhizosphere. Methods and Results: Hydroponic conditions: E. coli spray‐inoculated at log 4 CFU ml^?1 was recovered from leaf surfaces at a mean population of 1·6 log CFU g^?1 at 15 days. E. coli O157:H7 sprayed at log 2 or 4 CFU ml^?1 levelled off on spinach leaf surfaces at a mean average population of 1·4 log CFU g^?1 after 14 days, regardless of initial dose. Quantitative recovery was inconsistent across leaf developmental age. Field conditions: Average populations of E. coli O157:H7 spray‐inoculated at log 1·45 or 3·4 CFU m^?2 levelled off at log 1·2 CFU g^?1 over a 14‐day period. Pathogen recovery from leaves was inconsistent when compared to regularly positive detection on basal shoot tissue. Pathogen recovery from soil was inconsistent among sampling locations. Moisture content varied up to 40% DW and was associated with 50% (P < 0·05) decrease in positive locations for E. coli O157:H7 but not for E. coli. Conclusions: Overall, similar populations of environmental E. coli and E. coli O157:H7 were recovered from plants despite differences in inoculum dose and agronomic conditions. Strain source had a significant impact on the quantitative level and duration of survival on leaves and in soil. Water availability appeared to be the determinant factor in survival of E. coli and E. coli O157:H7; however, E. coli showed greater environmental fitness. Significance and Impact of the Study: Persistence of surrogate, indicator E. coli and E. coli O157:H7, irrespective of variable growing conditions in spinach is predominantly limited by water availability, strain source and localization within the plant. These findings are anticipated to ultimately be adopted into routine and investigative pathogen testing protocols and mechanical harvest practices of spinach.     

Actin-capping protein promotes microtubule stability by antagonizing the actin activity of mDia1

In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin–MT cross-talk in which a single factor acts sequentially on actin and MTs.     

Cutaneous Papilloma and Squamous Cell Carcinoma Therapy Utilizing Nanosecond Pulsed Electric Fields (nsPEF)

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7–30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.     

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.